A review of infectious interstitial and bronchointerstitial pneumonia in cattle with an algorithm for the detection of infectious and non-infectious causes

Abstract

Bovine interstitial and bronchointerstitial pneumonias are common and important diseases of cattle, caused by several infectious and non-infectious causes. Here, we review the roles of bovine respiratory syncytial virus, bovine parainfluenza virus 3, bovine alphaherpesvirus 1, bovine viral diarrhea virus, bovine coronavirus, influenza D virus, malignant catarrhal fever virus, and bovine adenovirus in interstitial or bronchointerstitial pneumonia. We describe the possible causes, pathogenesis, and diagnosis of bacterial septicemias that result in interstitial pneumonia, including E. coli, Salmonella, and Pasteurella multocida septicemias. We also review the parasitic causes of interstitial or bronchointerstitial pneumonia, primarily Dictyocaulus viviparus. Reaching a definitive postmortem etiologic diagnosis of interstitial or bronchointerstitial pneumonia can be challenging because infectious and non-infectious causes may look very similar grossly. Moreover, other conditions—that do not cause interstitial or bronchointerstitial pneumonia but rather pulmonary edema, congestion, and hemorrhage—can resemble interstitial pneumonia grossly. To guide the process of diagnosing interstitial and bronchointerstitial pneumonia, we offer an algorithm that integrates findings obtained from postmortem examination and ancillary laboratory testing. Our algorithm includes details on the gross characteristics of the lungs with interstitial or bronchointerstitial pneumonia, and we discuss other disease processes that may grossly resemble interstitial pneumonia. We highlight the key histologic features for differentiating specific causes and describe the most common ancillary laboratory tests to detect infectious and non-infectious causes.

Bovine interstitial pneumonia (IP) and bronchointerstitial pneumonia (BrIntP) are common and important diseases of cattle, with infectious and non-infectious causes.^15,27,127 The classification of pneumonia has been subject to much debate and no consensus exists regarding the terminology used to classify pneumonias in veterinary medicine.^26,96 Broadly speaking, based on the distribution and the type of lesion, pneumonias can be classified into 4 grossly distinct types: 1) bronchopneumonia (cranioventral), 2) interstitial (diffuse), 3) embolic (multifocal), and 4) granulomatous (focal or multifocal) pneumonia. ⁹⁶ Lung texture is also an important characteristic to consider when classifying pneumonias. In bronchopneumonia, the lungs are firm (often referred to as “consolidated”). In IP, lung texture is more elastic or rubbery (sometimes also referred to as “meaty”). In embolic and granulomatous pneumonias, the lungs feel nodular (nodules are firm and can vary in size). Identifying these 4 gross distributions allows the clinician or pathologist to narrow down possible causes, and guide sample collection and laboratory test requests. Note too that 2 or more gross types may be present simultaneously. ⁹⁶

A diffuse distribution of lung injury is arguably the most difficult distribution to interpret during the autopsy, as other pulmonary and non-pulmonary diseases can produce pulmonary congestion, edema, hemorrhage, hyperinflation, and emphysema, resulting in a morphologic distribution of lung disease that may be mistaken for IP.^23,27,96 We use IP to refer to pneumonia in which, grossly, the lungs are diffusely affected, either in a uniform or a lobular (also known as “checkerboard”) distribution. Histologically, the main injury to the lungs with a gross pattern of IP is in the alveolar or interlobular septa and is seen as 3 possible phases of diffuse alveolar damage (i.e., acute or exudative, subacute or proliferative, chronic or fibrotic).

BrIntP is a microscopic term (not a gross pattern) used when primary lesions in the bronchi and bronchioles are present along with alveolar damage. In cattle, BrIntP is frequently associated with viral infections, as many viral respiratory pathogens replicate and cause injury in respiratory epithelial cells of the bronchi, bronchioles, and alveolar septa.^23,27,50,96 Grossly, BrIntP can have a diffuse distribution similar to IP or a cranioventral distribution similar to bronchopneumonia. These patterns can lead to confusion, as the cranioventral distribution is widely associated with bacterial bronchopneumonia ⁹⁶ and less with viral BrIntP. However, this cranioventral distribution of viral BrIntP can be caused by bovine respiratory syncytial virus (BRSV; family Pneumoviridae, taxon species Orthopneumovirus bovis) and bovine parainfluenza virus 3 (BPIV3; family Paramyxoviridae, taxon species Respirovirus bovis). We will not discuss granulomatous or embolic pneumonia because their gross distributions differ from the diffuse gross distribution of IP just described.^23,96

The etiologic diagnosis of IP is often challenging given the considerable overlap in clinical signs and gross and microscopic lesions among the various infectious and non-infectious causes of IP.^23,27 Moreover, in some instances, the cause of IP or BrIntP remains unknown.^31,127 Infectious causes include viruses, bacteria, and parasites.^15,27,127 Non-infectious causes of IP that produce diffuse alveolar damage, and can cause herd outbreaks, are important differential diagnoses of infectious IP or BrIntP and include ingested toxins such as 3-methylindole (from lush forages),^21,71 4-ipomeanol (from moldy sweet potatoes), ⁴⁶ perilla ketone (from perilla mint), ⁶¹ paraquat (a toxic herbicide), Brassica species,^26,27 and Crotalaria mucronata. ¹³⁸ We will discuss these non-infectious causes in the context of the algorithm, but will not review them. We review common infectious causes of IP and of BrIntP and present an algorithm to guide the diagnostic investigation of cases of IP and BrIntP.

Our manuscript was structured to enable readers to consult it either in its entirety or by individual sections. Our review provides an updated summary of diagnostically relevant aspects of common infectious causes, whereas our algorithm functions as a stand-alone, practical framework for the diagnostic investigation of interstitial pneumonia in cattle, encompassing all of the most common possible causes.

Viral causes of IP or BrIntP

Viruses such as BRSV, BPIV3, and bovine alphaherpesvirus 1 (BoAHV1; infectious bovine rhinotracheitis virus [IBRV], family Orthoherpesviridae, taxon species Varicellovirus bovinealpha1) have long been recognized as important pathogens in the bovine respiratory disease complex (BRDC)—either as primary pathogens causing upper respiratory tract (URT) disease or BrIntP or as agents that predispose cattle to bacterial bronchopneumonia.^27,50,96,127 Other common viruses of cattle, such as bovine viral diarrhea virus (BVDV; family Flaviviridae, genus Pestivirus) and bovine coronavirus (BCoV; family Coronaviridae, taxon species Betacoronavirus 1) are typically associated with other disease syndromes, such as enteric disease (BVDV and BCoV), immunosuppression (BVDV), and reproductive disease (BVDV).^{27,113,168,177}

Debate continues regarding the importance of BVDV and BCoV as primary respiratory pathogens in cattle. However, these viruses may either predispose to BRDC, participate in BRDC as co-pathogens, or cause mild nonspecific lung lesions as part of a systemic infection (BVDV). Some authors suggest that BCoV may be a primary respiratory pathogen of cattle under certain circumstances.^{27,50,76,127,134,137,168} Influenza D virus (IDV; family Orthomyxoviridae, taxon species Deltainfluenzavirus influenzae) is prevalent in the URT of cattle, and both URT and lower respiratory tract (LRT) disease have been reproduced experimentally; however, the role of IDV in natural disease and in the BRDC is likely minor.^3,55,94,145 Another group of viruses, including the malignant catarrhal fever virus (MCFV) group and adenoviruses are not usually considered part of the BRDC but may cause systemic infections that involve the lungs.^115,124,172

Bovine alphaherpesvirus 1

BoAHV1 is one of the most important viral pathogens of beef and dairy cattle throughout the world.^13,88 This virus can cause a variety of conditions, including URT infections (infectious bovine rhinotracheitis), BrIntP, immunosuppression, keratoconjunctivitis, balanoposthitis, vulvovaginitis, systemic infections in bovine fetuses and young calves,^13,26,82 reduction in milk production, and abortions in pregnant cows. ⁸⁸ BoAHV1 transmission occurs through direct nose-to-nose contact, mucosal droplets from an infected animal, or aerogenously with exposure of mucosal surfaces to the virus.^88,93,100 Mating and artificial insemination from infected cattle are other possible transmission routes. The incubation period for the respiratory form is 2–6 d, and the virus can be shed in nasal secretions for 10–17 d.^93,104

BoAHV1 replicates in the epithelium of the URT and can undergo latency in neurons of sensory ganglia, most commonly the trigeminal ganglion, but also in other tissues, such as tonsils. ⁵⁰ Viral reactivation can occur during periods of stress and immunosuppression, such as during pregnancy and parturition and after transport or weaning. This reactivation is crucial for the maintenance of the virus within cattle herds.^82,88 During reactivation, the virus is transported to the initial infection site, where it initiates a new replication cycle and release of new virions. ⁸²

Clinical signs are largely the result of ocular and URT disease and include fever, anorexia, coughing, nasal discharge, drooling, conjunctivitis, keratoconjunctivitis, ocular discharge, hyperemic nares, and dyspnea. ⁸² Respiratory problems, abortions, or reproductive disease can occur during initial infection and reactivation periods. ⁸⁸ Abortions can occur at the same time or briefly after the respiratory disease. ⁸²

BoAHV1 causes mainly degeneration and necrosis in the respiratory epithelium of the nasal cavity, larynx, and trachea. It can also occasionally cause BrIntP, with damage to the bronchiolar epithelium and type I pneumocytes, and subsequent proliferation of type II pneumocytes. A primary viral IP can occur in young calves without lesions in the URT and without secondary bacterial bronchopneumonia. In these calves, the lungs can occasionally have epithelial syncytia in alveoli and, although not always present, characteristic eosinophilic intranuclear inclusion bodies (INIBs).^26,27 Bovine fetuses aborted as a result of infection by BoAHV1 develop the systemic form of the disease, which includes foci of acute necrosis and inflammation in the alveolar septa of the lungs, as well as necrotizing lesions in other organs, often with characteristic INIBs in cells at the margins of the necrotic areas. ¹¹²

For the diagnosis of BoAHV1 infection, it is important to consider both subclinical and clinical forms of infection. The subclinical form may lead to impaired immunity and predispose to secondary bacterial infections, such as bronchopneumonia. Therefore, it is important to test for BoAHV1 even in the absence of gross and microscopic lesions in the URT and LRT.^26,27 PCR testing of ocular or nasopharyngeal swabs (from live animals) or fresh, frozen URT or lung samples (from autopsied animals), and immunohistochemistry (IHC) from formalin-fixed, paraffin-embedded (FFPE) tissues, are useful for detecting BoAHV1 DNA or antigen, respectively.^27,129 If BoAHV1 antigen or DNA are detected by ancillary tests in the absence of gross and microscopic lesions, the result needs to be carefully interpreted, given that coinfections by other viruses or the stress of disease may induce reactivation of latent herpesviral infection. BoAHV1 vaccine viral DNA can be detected in transtracheal washes or nasopharyngeal swabs by PCR following intranasal or injectable vaccination using modified-live BoAHV1 vaccines. ¹⁷⁰ Virus isolation is infrequently used nowadays for routine testing, but the virus readily replicates in cell culture and produces characteristic cytopathic effects. ²⁶ To increase the chances of detecting BoAHV1 in tissues, samples should be collected in the early (acute) stages of the disease. If samples are collected later in the disease process, PCR and IHC test results may be negative. Therefore, the time of sampling should be considered when interpreting a negative test result. In live animals with respiratory problems, detection of seroconversion may associate BoAHV1 infection with clinical disease. Serum samples are more representative if collected in the acute and convalescent periods, typically 2 or 3 wk apart.^19,27,129

Bovine respiratory syncytial virus

First described in 1970, ¹²⁶ BRSV remains one of the main pathogens associated with the BRDC, and can cause severe BrIntP as a primary agent. ¹²⁹ This virus affects mainly young animals, but adult animals are also susceptible, especially if they have not been exposed previously. ⁵¹ In calves, the immunity generated is short-lived and reinfections can occur at any age, thereby maintaining viral circulation in the population.^8,16 The disease can take an acute form, leading to outbreaks in a limited time, or it can circulate continuously, causing intermittent disease with a wide range of morbidity and mortality rates across various management systems. ¹⁶² Although aerosols and direct contact between animals are the main transmission routes, spread by humans and fomites is also possible. ¹²² Most cases of BRSV infection are observed in autumn and winter, but infections can occur in any season. Infections by BRSV can be subclinical, affect only the URT, or involve both the URT and LRT.^11,51,162

Once in the host, BRSV targets ciliated and non-ciliated epithelial cells along the respiratory tract. The infection starts when the viral G protein binds to the cell surface. Entry into the cell cytoplasm is then mediated by the fusion (F) protein. ³⁸ The virus then replicates and is later released through budding directly from the apical membrane of epithelial cells, often inducing the epithelial syncytia characteristic of BRSV infection.^142,162 Disruption of the mucociliary function, from the loss of ciliated respiratory epithelial cells in BRSV-infected airways, can significantly contribute to the development of secondary bacterial bronchopneumonia. ⁵⁸

Clinical signs in young and adult animals are similar and include coughing, serous-to-mucoid nasal and ocular discharge, depression, anorexia, hyperthermia, and polypnea. Decreased milk yield can occur in lactating cows. ¹⁰² During lung auscultation, abnormal breathing sounds may be detected. In severe cases, infected animals can develop pneumothorax and assume an orthopneic position, with neck and head extended and the elbows spread outward. When animals with severe pulmonary emphysema survive for 24–72 h (personal observation), the emphysema can extend to the subcutaneous tissues.^27,162 This is thought to occur as air from the interstitial pulmonary emphysema extends along fascial planes of the mediastinum to the subcutis. ²⁶

At autopsy, variations in the distribution and severity of gross lesions occur. Although BrIntP is a microscopic pattern, BrIntP caused by BRSV can appear grossly as a well-demarcated area of cranioventral consolidation. The affected area is dark pink-to-red and atelectatic; caudodorsal areas of the lungs can be normal or sometimes distended, rubbery, lighter in color, and emphysematous. ¹⁴² This distribution of lesions can be similar to the gross appearance of bacterial bronchopneumonia, and differentiation can be challenging at the gross level. Moreover, the cranioventral distribution of consolidation in BRSV BrIntP can sometimes be concurrent with secondary bacterial bronchopneumonia. A diffuse uniform or lobular distribution of lung injury associated with BRSV infection is also possible. In this distribution, both lungs are diffusely expanded, rubbery, with variable intra-alveolar and interlobular septal edema, emphysema, and pleural hemorrhage. Microscopically, in the early stages of disease, BrIntP occurs, with formation of syncytia within the bronchiolar and alveolar epithelium and eosinophilic intracytoplasmic inclusion bodies (ICIBs) within respiratory epithelium and syncytial cells.^23,26,96

Direct and indirect laboratory tests are available for the detection of BRSV infection in tissues and serum samples. Fresh samples for real-time PCR (rtPCR) testing or antigen detection by a direct fluorescent antibody (DFA) test include nasal swabs, deep nasal or nasopharyngeal swabs, transtracheal washes, bronchoalveolar lavages, and, from the dead animal, lung tissue. ¹²⁹ Respiratory viruses, including BRSV, are only shed for a limited time. Therefore, to increase the chances of detecting BRSV, sampling of animals in the acute stage of the disease is recommended and, if possible, sampling of more than one animal. Following intranasal vaccination with live vaccines, vaccine viral RNA can be detected up to 14 d post-vaccination in nasal swabs or transtracheal washes via rtPCR in a high proportion of animals.^158,170 Vaccine viral RNA can be detected in the transtracheal wash of ~10% of calves by rtPCR after injectable vaccination with multivalent modified-live viral vaccines that include BRSV. ¹⁷⁰ Therefore, recent vaccination history and vaccination route must be considered when interpreting positive rtPCR results. Sequencing can be performed to differentiate between vaccine strains and wild-type virus. This approach allows identification of genetic differences between vaccine-derived and field strains.^59,60 Virus isolation is not recommended for the diagnosis of BRSV infection because the immune response generated by the host interferes with the test and the virus is easily inactivated in transport. ²⁶

Antibodies against BRSV can be detected by several commercial ELISAs, which are cost-effective and enable the analysis of multiple samples in a few hours. Positive serum samples collected 5–10 d after the onset of clinical signs can indicate recent BRSV infection, provided the animals were neither vaccinated nor exposed previously. However, because the virus is widespread in cattle populations, antibodies may be present—from maternally derived immunity, prior vaccination, or previous exposure to field strains. Thus, serologic results must be interpreted with caution. Paired serology comparing acute and convalescent samples to demonstrate seroconversion is preferable to demonstration of acute infection. ⁸⁷ Milk samples are also suitable for serologic testing to monitor viral circulation in the herd. Although antibody titers tend to be lower in milk than in serum, correlation is generally good between milk and serum. ¹²¹ Histopathology is often a very helpful postmortem ancillary test, as the virus causes characteristic microscopic lesions in the lungs, especially in the acute phase of infection. The finding of typical microscopic lesions in the lungs also facilitates the interpretation of positive rtPCR results, because lesions are usually present only in natural infection and absent in recently vaccinated animals. When histologic lesions are suggestive of BRSV infection, viral antigen in the lesions can then be investigated via IHC on FFPE tissue.^26,27,96,129

Bovine parainfluenza virus 3

BPIV3 virus has been associated with the BRDC since its first isolation in the United States in 1959. ¹³⁵ Subsequently, the endemic presence of BPIV3 in cattle populations worldwide has been proven through serosurveys.^{48,56,85,91,105} The virus can be detected serologically in clinically normal cattle populations; therefore, detection of antibodies does not necessarily indicate an association with the BRDC. ¹⁷⁴ BPIV3 spreads via direct contact and aerosols. Infections typically occur in the autumn and winter months in temperate climates but, as with BRSV infection, they can happen in any season. Most reported natural and experimental infections have occurred in young animals, 2–8-mo-old. The duration of immunity following BPIV3 infection is unclear. However, experimental studies have shown that seropositive calves can be reinfected as early as a few weeks after initial exposure and potentially shed the virus again, although typically for a shorter time than during primary infection. ⁴⁹ Even though clinical disease associated with BPIV3 is infrequent, the virus is considered a primary respiratory pathogen and is able to damage the respiratory tract independent of other pathogens.^84,129

BPIV3 is found in the nasal passages, trachea, and bronchiolar and alveolar epithelial cells. In the respiratory tract, the virus binds to sialic acid receptors on host epithelial cells through the hemagglutinin-neuraminidase surface protein; cell entry is then facilitated by the F protein. ¹⁰² BPIV3 infects ciliated and non-ciliated epithelial cells of the URT and LRT, alveolar macrophages, type II pneumocytes, and lymphocytes. The main role of BPIV3 is the reduction of respiratory defenses, which predisposes animals to secondary bacterial infections. One of the primary mechanisms identified is the reduction of ciliated epithelial activity, which results in impaired mucociliary clearance. In addition, the virus compromises several critical functions of alveolar macrophages, including Fc receptor expression, phagocytosis, and microbicidal activity. ⁴⁹

Clinical signs associated with BPIV3 primary disease include fever, cough, nasal and ocular discharge, tachypnea, and increased lung sounds and wheezes. ⁸⁴ If present, the gross findings in natural cases are usually mild and may be localized to the cranioventral regions or have a lobular distribution affecting any of the lung lobes. ² The lungs can be rubbery or, if secondary bacterial infection occurs, firm or consolidated. Microscopically, in natural cases of BPIV3 infection, BrIntP with bronchiolar degeneration and necrosis (and characteristic eosinophilic ICIBs and INIBs in various cell types) are common in the early stages of disease. Epithelial syncytial cells may also be present in the alveoli in some instances. Additionally, the walls of the bronchioles and the alveoli are often edematous, accompanied by increased macrophages and neutrophils, along with proliferation of type II pneumocytes.^26,27,49

Preferred samples for virus isolation, rtPCR, or antigen-based tests such as DFA, include nasal swabs, deep nasal swabs, transtracheal washes, bronchoalveolar lavages, and, from the dead animal, lung tissue. Similar to BRSV, BPIV3 is only shed for a limited time, and sample collection during the early stages of infection is key to identifying viral infection. IHC can be performed on FFPE tissues.^27,35,129 Subacute cases or cases with secondary bacterial bronchopneumonia may yield negative BPIV3 test results.^27,129 Serologic tests, such as ELISA, are also available to detect antibodies against BPIV3. In a clinical setting, sampling in the early stages of viral infection can be complicated, which may underestimate the true prevalence of BPIV3-associated disease. To accurately interpret serologic tests, it is important to collect samples from multiple animals during both the acute and convalescent periods of the disease (2 or 3 wk apart).^27,35,129 Because of the endemic nature of BPIV3, maternal antibodies and vaccination can complicate the interpretation of results. Additionally, some animals may not develop a serologic response after infection. ELISA serology is still considered a valuable tool for epidemiologic studies and for monitoring herd health status. ⁴⁹

Bovine viral diarrhea virus

BVDV is an important viral pathogen in cattle. There are 2 primary genotypes: BVDV1 (taxon species Pestivirus bovis) and BVDV2 (taxon species Pestivirus tauri), each of them having cytopathic (CP) and noncytopathic (NCP) biotypes.^101,177 The virus can infect cattle of all ages, including fetuses. It is considered an important agent in the BRDC, mainly because of its ability to suppress the immune system, which predisposes cattle to coinfection with other respiratory viruses and bacteria.^{12,62,90,133,177} Although some studies suggest that BVDV may cause mild clinical respiratory signs (such as nasal discharge and coughing),^90,133,177 the role of BVDV as a primary pneumonia pathogen in cattle has not been confirmed.

During acute BVDV infection, viral antigen can be demonstrated in alveolar macrophages and airway epithelium in the lungs and in the muscularis of arterioles in the lung, heart, and other tissues. This indicates wide tissue tropism by the virus, but not necessarily primary tissue damage or clinical disease, given that there may be no histologic lesions.^12,26

The virus can cause a wide range of clinical and pathology presentations, including subclinical infections, fever, nasal discharge, coughing, lameness caused by ulcerative interdigital dermatitis and coronitis, diarrhea, erosive and ulcerative enteritis (mucosal disease), hemorrhagic syndrome, embryonic and fetal deaths, and fetal malformations. When bovine fetuses are exposed to the virus before they develop immune competence, they can be born persistently infected (PI). These PI calves are a very important environmental source of the virus, maintaining virus circulation and spread within and among cattle herds.^62,95,177

There are typically no gross pulmonary lesions in BVDV-infected cattle. Histologic lesions in the lung are also infrequent, mild, and nonspecific, including peribronchiolar, perivascular, or interstitial aggregates of lymphocytes and macrophages, and/or mild suppurative exudate in bronchioles.^26,133 The virus can be detected in the lungs of cattle succumbing to the BRDC, but the pathology changes observed are usually attributed to other viral or bacterial agents of the BRDC and not to BVDV alone. ¹²

Given that BVDV is considered an important predisposing agent for the development of the BRDC, it is often included in test panels offered at reference laboratories. A wide variety of laboratory tests can detect BVDV, including virus isolation, serology, antigen detection in tissues (DFA, IHC in FFPE tissues), rtPCR, and others. Control programs that incorporate serology, antigen ELISA, IHC, or pooled rtPCR testing have successfully reduced the prevalence of PI cattle. Interpretation of these results must account for factors such as vaccination history, transient infections (animals may be positive but are not PI), and age at testing (colostral antibodies may interfere with some test results). Pooled rtPCR testing is efficient for screening, but follow-up individual testing is necessary to identify PI animals.^171,175,177

Bovine coronavirus

BCoV is widespread throughout the world as a result of rapid viral transmission and the presence of healthy carrier animals in the cattle population. ¹⁸⁰ The virus is widely recognized as an enteric pathogen, causing diarrhea and enterocolitis in calves and adult cattle. Less commonly, BCoV has been associated with respiratory infections, and occasionally respiratory disease, in cattle of all ages.^76,143,150 Although evidence supporting the role of BCoV in respiratory infections is increasing,^134,150 its role in the BRDC is still under debate because the virus has been detected in both sick and healthy animals and experimental replication of respiratory disease with BCoV has been difficult.^{44,76,143,168}

The transmission of BCoV is primarily through the fecal-oral route, but aerosol transmission is also a likely route of infection.^{76,123,150,168} The virus infects epithelial cells in the respiratory tract and the intestines. Viral replication leads to shedding in nasal secretions and in feces; nasal shedding often precedes fecal shedding.^123,157 Although further investigations are needed, some authors believe the initial replication occurs in the respiratory tract and then reaches the gastrointestinal tract through ingestion of mucus with viral particles.^76,123,143 Other authors believe that the intestinal tract is infected first and the virus then reaches the respiratory tract through viremia.^14,76,130 The virus can be shed in feces and nasal secretions for several months after infection.^32,83,130 This persistent shedding is critical for virus circulation in the cattle population. Cow-to-calf and calf-to-calf transmission are the most common routes of infection. ⁷⁶

In the experimental respiratory form of BCoV disease, mild respiratory disease or pneumonia, mainly in calves up to 6-mo-old, have been described.^123,150,168 Clinical signs include nasal secretions, dyspnea, coughing, and fever.^{76,123,143,150} The respiratory lesions associated with BCoV infection are bronchial and bronchiolar epithelial degeneration and necrosis, ²⁶ but the respiratory lesions associated with natural BCoV respiratory disease are not well reported in the literature.

For the diagnosis of BCoV infection in cases of respiratory disease, rtPCR is useful to detect viral RNA in nasal swabs, nasopharyngeal swabs, or URT tissues and lung. ¹²³ IHC and in situ hybridization (ISH) can be used to detect viral antigen and RNA in FFPE tissue sections with lesions, respectively. In one study, ISH was significantly more sensitive than IHC in detecting the virus in the trachea and lung. ¹³⁴ Detection of BCoV antibodies in serum can assess exposure or seroprevalence in a given population, but serology can be very difficult to interpret as an indicator of acute BCoV disease. ⁷⁶

Influenza D virus

IDVs are known to cause respiratory disease in a wide range of hosts. Historically, influenza has not impacted cattle as significantly as it has other species. ¹⁵³ Only natural sporadic outbreaks of respiratory disease, mainly related to influenza A virus, and a few experimental studies can be found in relation to cattle.^97,153,155 Recent outbreaks of highly pathogenic avian influenza (HPAI) A(H5N1) virus in the United States have shown that cattle are less resistant to influenza viruses than previously thought. In these outbreaks, cattle had decreased feed intake, mild respiratory signs (clear nasal discharge, tachypnea, and dyspnea), lethargy, dry or tacky feces, diarrhea, mammary gland involution, decreased milk production, and milk with abnormal color and consistency.^25,89,152 To date, interstitial pneumonia has not been reported in cattle infected with HPAI A(H5N1). IDV-specific antibodies have been detected in several species, including humans. However, the presence of IDV in cattle from regions around the world and the higher titers in cattle compared with other species suggest that cattle are the primary reservoir for IDV. ⁶⁹

Despite the widespread presence of IDV antibodies in the cattle population, its role and impact on BRD are not fully understood, especially under field conditions. Molecular surveillance studies, using nasal swabs of cattle with respiratory clinical signs, suggest that IDV may have a role in the BRDC.^{30,34,111,116,141} However, IDV has also been identified in healthy cattle and some authors have even suggested that IDV may be protective, reducing the severity of the BRDC.^54,110,144 In addition, higher or equal viral loads in subclinical cattle, compared with those with respiratory clinical signs, have been reported. As long as the immune defense mechanisms of cattle are stable, IDV does not cause respiratory disease. However, healthy subclinical cattle can be carriers and help to spread the virus. ⁵⁵ More studies need to be conducted to better determine the role of IDV in BRD under natural conditions. Under experimental conditions, infected calves developed mild-to-moderate respiratory disease.^55,145

The URT is primarily targeted by IDV, but the virus can also reach the LRT. The virus has been detected in the nasal cavity, trachea, bronchioles, and the cranial, middle, accessory, and caudal lung lobes, as well as in tracheobronchial and mediastinal lymph nodes. The highest RNA load is found in the nasal cavity, with significant levels also noted in the olfactory bulb and tonsils of sentinel animals exposed via aerosol.^55,94,145

Experimental studies have also been conducted to investigate the effect of IDV as a co-pathogen. Complex interactions and outcomes from coinfections with IDV and other pathogens, such as BVDV, Mycoplasmopsis (Mycoplasma) bovis, and Mannheimia haemolytica have been observed.^94,167,179 It is beyond the scope of our review to address these interactions, and readers are encouraged to review the original literature.^4,94,167,179

Clinical signs peak at ~8 d post infection (dpi), and include dry cough, nasal and ocular discharge, depression, and mild-to-moderate tachypnea. In more severe cases, dyspnea and abnormal lung sounds, including wheezing, may occur.^55,94,145

Lesions, if present, are mainly restricted to the LRT, and areas of mild IP are commonly located in the right cranial lung lobe.^54,145 Microscopically, rhinitis and tracheitis are found, sometimes with deciliation, erosion, and necrosis of respiratory epithelium. Occasionally, there can be interstitial or BrIntP. ¹⁴⁵

The detection of IDV infection and identification of the virus rely on laboratory tests, including virus isolation, rtPCR (viral RNA), IHC (viral antigen), and serology (antibodies against IDV).^53,70,114 Nasal swabs are commonly used for sampling because they offer a high yield for virus detection. Bronchoalveolar lavage fluids, transtracheal washes, or lung tissue from autopsy can also be used for molecular detection. ³ In Sweden, bulk tank milk samples were tested using an in-house indirect ELISA, ⁵ and cow milk was suggested as an alternative sample in IDV surveillance. IHC on FFPE lung or other tissue sections can be used to identify IDV antigen.^55,145

Other viruses occasionally associated with bovine IP

Other viruses, including the MCFV group and bovine adenovirus, occasionally can cause IP. Neither of these viruses are considered key players in the BRDC.^115,124,172

Malignant catarrhal fever virus group

Of the several herpesviruses that have been included in this group, 2 are more commonly associated with disease in cattle—alcelaphine gammaherpesvirus 1 (AlGHV1; Orthoherpesviridae, Macavirus alcelaphinegamma1) and ovine gammaherpesvirus 2 (OvGHV2; Macavirus ovinegamma2). Malignant catarrhal fever (MCF) is a severe, often fatal, systemic disease of cattle caused by these viruses. The pathogenesis of MCF is mainly associated with lymphoproliferation and vasculitis, which can be observed in multiple organs.

Clinical signs vary, but include fever, lethargy, ocular and nasal discharge, and corneal opacity (panophthalmitis). The characteristic lesions are generalized lymphadenopathy and inflammation and necrosis in the alimentary, upper respiratory, and urinary tracts. Dermatitis, encephalitis, and arthritis can also occur.^74,124 The microscopic lesions observed in cattle with clinical MCF include vasculitis (predominantly arteritis) of mid-caliber vessels, lymphoid hyperplasia, and mucosal necrosis and ulceration in any portion of the alimentary tract and URT. In the lungs, as in other organs, necrotizing vasculitis occurs, which may be accompanied by lymphoplasmacytic interstitial inflammation.^{26,74,124,146}

A definitive diagnosis of MCF requires a combination of appropriate epidemiology, clinical signs, gross and histologic lesions, and detection of any of AlGHV1 or OvGHV2 DNA (PCR) or antigens (IHC) in fresh or FFPE tissue, respectively. ¹²⁴

Bovine adenovirus

BAdV (family Adenoviridae, genus Mastadenovirus) can sporadically cause respiratory, gastrointestinal, and ocular disease in cattle, especially in young or immunocompromised animals. Respiratory adenoviral infection has been experimentally reproduced in calves deprived of colostrum and infected with bovine adenovirus 3 (BAdV3; Mastadenovirus bostertium), ⁴⁰ as well as in calves treated with dexamethasone prior to endobronchial inoculation of BAdV3. ¹¹⁵ The severity of primary adenoviral disease, including pneumonia, is probably dependent on the virus strain and the immune status of the calf. ²⁶ BAdV can be isolated occasionally from calves with bacterial pneumonia in feedlots, and seroconversion occurs within a few weeks of entering the feedlot. However, the importance of BAdV as a contributor in the BRDC is probably minor.

Lesions associated with primary BAdV infection in the lungs are well-demarcated, lobular or patchy areas of atelectasis and reddening. Histologically, the acute stage of the disease features degeneration, necrosis, and regeneration of the bronchial and bronchiolar epithelium, lymphocytic infiltration of airway epithelium, and formation of large basophilic INIBs. The acute lesions can progress to extensive bronchiolar necrosis and increased numbers of mononuclear cells in the alveolar septa. Pneumocyte type II hyperplasia, a feature of other causes of IP, is uncommon.^26,96,115

The diagnosis of adenoviral pneumonia begins by finding histologic lesions compatible with the characteristic viral INIBs in lung sections, which can be followed by identification of viral DNA (PCR) or antigen in fresh (DFA) or FFPE tissues (IHC). ²⁶

Bacterial causes of IP

Pathogenic bacteria and their toxins disseminate via the bloodstream, enter and exit through the endothelium, and trigger a cascade of pathologic events that contribute to vascular inflammation.^96,165 The endothelium of blood vessels is a critical barrier between the blood and underlying tissues, regulating permeability, inflammation, and homeostasis. In cattle, bacteria (such as Salmonella, Escherichia coli, and Pasteurella multocida) can cause sepsis and exploit lung endothelial vulnerabilities, leading to increased vascular permeability and interstitial pneumonia via diffuse alveolar damage. ²³ We focus on the lesions induced by these 3 agents, acknowledging that other bacteria entering the circulation may cause similar lesions.

Pathogenesis of bacterial IP

The lung is uniquely susceptible to bacterial infection because of the dual exposure routes that permit entry of bacterial organisms: aerogenous and hematogenous. The aerogenous route of bacterial infection is frequently associated with bronchopneumonia, which is beyond the scope of our review. The hematogenous route of infection is associated with interstitial lung lesions. The portals of entry of bacteria into the blood include skin wounds, surgical wounds, neonatal umbilicus, mammae, and invasion via any mucosal surface (the respiratory, ocular, alimentary, and reproductive mucosae). ¹⁵⁴ The primary damage is incurred to the alveolar septa, especially to alveolar type I pneumocytes and vascular endothelial cells.^23,26,96,127

Bacteria can damage endothelial cells via direct contact, generation of toxins, and/or the release of large amounts of cytokines from a local or distal site of infection (cytokinemia or “cytokine storm”). In direct damage, bacteria target specific vascular beds and interfere with endothelial homeostasis, weakening cellular junction stability and promoting endothelial cell proliferation and inflammation. ¹²⁰ Bacteria adhere to the endothelium, smooth muscle cells, or extracellular matrix, where they manipulate the host-cell actin cytoskeleton and induce internalization and transcytosis. Bacteria can also remain extracellular, disrupting the assembly of intercellular junctions to facilitate paracellular passage. These interactions can compromise vascular integrity, leading to endothelial dysfunction and enabling bacterial dissemination into surrounding tissues. ¹⁶¹

Two types of bacterial toxins may be involved in lung injury, exotoxins and endotoxins. Exotoxins, produced and secreted by gram-positive and gram-negative bacteria, may trigger alveolar epithelial and capillary endothelial cell death. ⁹⁸ The result is increased vascular permeability and edema, loss of epithelial cells, loss of ability to drain fluid from the alveoli, and, in more chronic stages, fibroplasia and fibrosis.^66,99 Endotoxins are glycolipids found in the cell wall of gram-negative bacteria, the most important of which are lipopolysaccharides (LPSs). Endotoxins are released upon bacterial death, triggering the production of pro-inflammatory cytokines, such as TNF and IL6.^86,98,176 This results in altered endothelial permeability, leukocyte movement between blood and tissues, and activation of the coagulation pathway.^86,96 LPS-induced oxidative stress also causes endothelial cell apoptosis. ¹¹⁸ The interstitial inflammation that results from bacterial or exotoxin- or endotoxin-mediated damage to the endothelium results in lung edema, capillary thrombosis, fibrin deposition in alveoli, and neutrophilic infiltration.^26,148,165 In addition to endothelial cell damage, sepsis can directly damage surfactant-producing type II pneumocytes, which, along with the serum proteins present in the exudate, contribute to surfactant dysfunction. This leads to atelectasis and abnormal surface tension in the alveoli, ultimately resulting in injury to type I pneumocytes.^26,79 Pulmonary endothelial cells have a unique sensitivity to LPS.^15,28

Cytokinemia is a dysregulated and excessive release of proinflammatory cytokines into the bloodstream, surpassing the level required for effective immune control. This phenomenon reflects a disproportionate or sustained activation of the immune system, leading to elevated concentrations of cytokines such as TNF, IL6, IL1β, and IFNγ. These cytokines mediate a wide range of effects, including cellular proliferation, differentiation, and autocrine, paracrine, and systemic signaling.^75,139

Pathology of bacterial IP

Bacterial septicemia or sepsis appears as a diffuse (and usually uniform) pattern of IP at autopsy. The lungs are diffusely expanded, dark-pink or red, heavy, wet, and rubbery; they fail to collapse and may bulge on cut sections. The cranioventral gross distribution of BrIntP observed in some viral infections (BRSV or BPIV3) is not observed with bacterial septicemia or sepsis.^{7,24,26,65,96,149}

Septicemic IP can manifest histologically in different ways. Severity and chronicity play a role in the different manifestations, but other unclear reasons may also contribute. One manifestation is thickening of alveolar septa, which are hypercellular because of infiltrates of neutrophils and mononuclear cells, or because of hypertrophy of pulmonary intravascular macrophages.^26,149 A second manifestation includes marked congestion of alveolar septa and interstitium, with serofibrinous, flocculent or fibrillar, eosinophilic exudate in the alveoli. Because of endothelial damage, acute intra-alveolar hemorrhage can also be present. Capillary or small venule thrombosis and the presence of bacterial emboli and bacteria within alveoli can occur. Occasionally, some cases may have diffuse alveolar damage, with hyaline membrane formation and type II pneumocyte hyperplasia as characteristic features.^{23,24,26,31,65,77,131}

Escherichia coli septicemia

The gram-negative bacterium E. coli includes a heterogeneous group of normal inhabitants of the gastrointestinal tract in cattle. According to plasmid-mediated virulence attributes, there are 2 key pathotypes of E coli, diarrheagenic and extraintestinal. ¹⁰³ Precipitating factors, such as viral infection, failure of passive transfer, nutritional imbalances, immunosuppression, and suboptimal environmental conditions, can promote opportunistic disease and the development of septicemic colibacillosis, leading to bacterial IP.^1,63

In neonatal calves, hematogenous dissemination of some E. coli pathotypes commonly occurs through ascending umbilical infections or secondary to neonatal calf diarrhea. ¹⁶³ Septicemic colibacillosis is a leading cause of death in neonatal calves. ¹¹⁸ E. coli endotoxemia occurs in adult cattle, particularly in cows with coliform mastitis. ⁶³

Although no pathognomonic features of IP are associated with E. coli, diagnosis can be achieved by corroborating history, gross findings, and histopathology, in combination with conventional bacterial cultures, serologic identification of the isolated strain, molecular identification of virulence factors, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS).^9,20,103

Salmonella spp. septicemia

Salmonella enterica subsp. enterica is an important gram-negative bacterial pathogen of cattle. ⁷⁸ Of the Salmonella serovars characterized by antigenic composition, most clinical cattle isolates are identified by their O (somatic) antigens and further subdivided into serogroups. Most isolates in cattle belong to either the non–host-specific serogroups B, C, and E, or to the host-adapted serovar, type D, which includes Salmonella Dublin. Salmonella Dublin affects cattle of all ages and is predominantly associated with septicemia and IP in calves and, occasionally, adult cattle.^10,117,131

Transmission occurs through fecal-oral contamination from infected livestock, rodents, and birds, feed contaminated with animal byproduct protein sources, or through aerosolization between closely confined calves.^67,78 Entry through the respiratory tract or ocular mucous membranes is uncommon. Infection may also occur in utero or through the mammary gland. In both calves and adults, the severity of disease is determined by serovar virulence, inoculum dose, immune status, previous exposure, age, and other stressors. ⁷⁸ Following intestinal colonization, Salmonella organisms invade the intestinal mucosa and can enter macrophages, which facilitates dissemination through the lymphatics and blood to extra-intestinal sites, including the lung. ¹¹⁷ In addition to Salmonella plasmid virulence (spy) genes, LPS is key to the pathogenesis of Salmonella Dublin infections. In a study across dairy cattle farms in British Columbia from 2007–2021, in cases in which Salmonella Dublin was the primary cause of morbidity and mortality, respiratory signs were most common as a result of sepsis-induced IP. ¹⁷ Other lesions that can be observed simultaneously with Salmonella Dublin septicemia and IP in calves include icterus, splenomegaly, and pleural or abdominal effusions, ¹⁶⁰

The diagnosis of Salmonella septicemia can be achieved by a combination of clinical history, gross findings, histopathology, conventional culture with serotyping, PCR, MALDI-TOF MS, or ELISA to assess antibody levels against O-antigens in blood and milk samples.^65,78,117

Pasteurella multocida septicemia

Members of the Pasteurellaceae family have frequently been associated with bronchopneumonia and septicemia. M. haemolytica is a well-established pathogen in the BRDC and is frequently implicated in progression to septicemia or sepsis. ³³ Bibersteinia trehalosi was documented as the main pathogen in an outbreak of fibrinous pleuropneumonia and systemic infection in newborn calves. ¹⁸ P. multocida, specifically serotype A:3, is frequently associated with the BRDC, and is commonly isolated from the lungs in cases of bronchopneumonia. ³⁹ However, bronchopneumonia caused by P. multocida should not be confused with hemorrhagic septicemia (or septicemic pasteurellosis) of cattle and water buffalo, which is caused by the inhalation or ingestion of serotypes 6:B and 6:E of P. multocida. ⁹²

Animals with hemorrhagic septicemia develop a high fever and die rapidly, with a 100% fatality rate. P. multocida is probably transmitted by ingestion or inhalation during direct contact with an infected animal or contaminated water. ¹⁴⁷ Other sources include decomposing carcasses and damp soil. Moreover, healthy cattle and wildlife species, such as elk and water buffaloes, can act as a reservoir for the bacterium in the environment. ⁴²

The key virulence factors for P. multocida are LPS, the capsule, and outer membrane proteins. The capsule is involved in bacterial resistance to phagocytosis and complement; LPS is critical for bacterial replication and survival within the host. ⁶⁸ Particularly in cases of hemorrhagic septicemia, the outer membrane protein H (OmpH) is critical to the pathogenicity and invasiveness of the bacterium. ⁸¹

Lesions of hemorrhagic septicemia include subcutaneous edema of the head, neck, and abdomen, pericarditis, hemorrhagic enteritis, hemorrhagic lymphadenitis in superficial and visceral lymph nodes, and necrotizing myositis.^47,96,147 In the thorax, gross findings include pleural effusion, edema, and hemorrhages, with or without consolidation of the pulmonary parenchyma. ⁹⁶ Histologically, lung lesions depend on the exposure route. Experimental aerosol exposure resulted in suppurative bronchopneumonia. ¹³⁸ Bacteria were numerous and frequently seen in lymphatic vessels. Intranasal and intramuscular exposure resulted in IP, with increased numbers of lymphocytes and macrophages in alveolar septa. Interestingly, cellular and vascular changes were more often present in the cranioventral aspect of the lung. ¹³⁶ A definitive diagnosis of hemorrhagic septicemia is reached by bacterial isolation and molecular detection of capsular antigen group P. multocida from the blood and tissues of an animal with the typical gross and histologic findings. ⁴⁷ Clinically affected animals die quickly, and serologic assays are usually not useful for the diagnosis.

Parasitic causes of IP

Dictyocaulus viviparus is the most important parasitic respiratory pathogen of cattle worldwide.^96,125,128 It can cause IP in cattle, affecting respiratory function and leading to impaired productivity, economic losses, and animal welfare concerns.^28,127 Infrequently, some gastrointestinal helminths, such as Ascaris suum and Toxocara vitulorum, can migrate through the lungs and cause IP.

Dictyocaulus viviparus

The geographic distribution of D. viviparus is mainly influenced by environmental conditions, with greater prevalence in temperate regions with high humidity and rainfall that favor larval survival on pasture.^43,106,128 A significant epidemiologic feature of D. viviparus is its association with Pilobolus fungi. The fungus grows in cattle dung, and larvae may accumulate on the sporangia of Pilobolus. Explosion of sporangia to disperse fungal spores also propels infective larvae onto surrounding vegetation, enhancing transmission efficiency.^106,128 The life cycle is direct. Adult worms reside in bronchi and lay eggs that hatch into first-stage larvae (L1) that are expelled via coughing and are swallowed before being passed in feces. L1 larvae develop within cow manure in the environment, becoming infective third-stage larvae (L3) in 5–7 d under favorable warm, moist conditions.^6,128 Ensheathed L3 larvae are infective to grazing cattle. Once ingested, L3 larvae exsheath in the abomasum and then reach the small intestine. There, the larvae penetrate the mucosa and migrate to the mesenteric lymph nodes where they remain for 3–8 d. Fourth-stage larvae (L4) reach the lungs as early as 7 dpi and can be present in large numbers at 15 dpi. The prepatent period is usually 3–4 wk. ¹²⁸ Although usually considered a disease of first-season grazers, dictyocaulosis can also affect adult cattle, especially dairy cows.^41,125,164 Immunity to D. viviparus develops after exposure, but wanes in the absence of reinfection. Outbreaks are more frequent in animals that have not been exposed previously to the parasite.^132,164

The pathogenicity of D. viviparus is largely dependent on the parasite burden and the immune status of the host. Low worm burdens are usually subclinical, and high worm burdens lead to extensive damage to the respiratory system, inducing inflammation, edema, and compromised lung function. ¹²⁸ The clinical signs and lesions differ between the different phases of the disease. During the prepatent period (7–25 dpi), animals cough and have tachypnea^127,128 as larvae migrate through the lungs, causing mechanical damage to the alveolar septa and inflammation. Grossly, the lungs can have a diffuse-uniform or diffuse-lobular distribution of interstitial pneumonia. During the prepatent period, parasites are often not detectable and, therefore, the lesions in this stage can mimic viral or toxic causes of BrIntP and IP, respectively. Larval migration induces alveolitis and bronchiolitis, IP, edema, and hemorrhage. Lesions include infiltration of macrophages, neutrophils, and eosinophils around bronchioles and within alveoli, thickening of alveolar septa, and increased vascular permeability. Parasite larvae may be observed in the alveoli or in terminal or respiratory bronchioles. ¹²⁸

The patent period (25–55 dpi) is associated with maturation and reproduction of adult worms in the bronchi and trachea and L1 larvae in the bronchi, bronchioles, and alveoli. Clinical signs during this period include dyspnea, coughing, pyrexia, loss of condition, stridor, and emphysematous crackling. As the stage of dictyocaulosis progresses from late-prepatent to patent periods and beyond, nodules or even areas of lobular consolidation may be present as a result of granuloma formation and large numbers of L1 larvae and eggs in bronchioles and alveoli. Adult nematodes can be found in the airways admixed with a variable amount of mucus, which is diagnostic for pulmonary dictyocaulosis. Histologically, damage to the respiratory epithelium of the trachea, bronchi, and bronchioles results in tracheitis and bronchitis. The airway also may be obstructed by large numbers of adult worms. Interstitial pneumonia, atelectasis, emphysema, and pulmonary edema can develop and contribute to the severity of clinical signs. Neutrophils, eosinophils, and mononuclear cells in variable numbers are observed within alveoli. Nodular lesions may be present as a result of focal inflammation or granuloma formation around dead larvae. Partial airway obstruction can cause lung collapse and emphysema in adjacent areas.^{22,125,127,128}

During the post-patent period, when adult worms die and are expelled, the host experiences a gradual decrease in the number of worms in the bronchi and trachea. Chronic lesions, such as peribronchial fibrosis, obliterative bronchiolitis, damage to alveolar septa, emphysema, and inflammation, may persist for weeks, contributing to prolonged coughing and increased susceptibility to secondary bacterial infections. Survivors with residual pulmonary fibrosis and chronic bronchitis may have variably severe long-term pulmonary dysfunction. The degree of recovery depends on the severity of the initial infection, possible reinfections, and the host immune status.^127,128

Diagnosis of dictyocaulosis in most cases is based on knowledge of local parasite epidemiology, clinical history, clinical signs, and gross pathology. Demonstration of L1 larvae in fresh fecal material, or nematodes in the airways during autopsy, confirms the diagnosis. L1 larvae can be detected in feces using the Baermann laboratory technique. ¹²⁸ A PCR assay may be used to detect dead or viable larvae of D. viviparus in individual or pooled fecal samples. ¹⁶⁹

Other helminths

Infrequently, some gastrointestinal helminths, such as A. suum and T. vitulorum, can also cause IP while migrating through the lungs. A. suum, a nematode primarily infecting swine, is a possible cause of pulmonary disease in cattle, particularly in environments in which calves are exposed to contaminated swine feces.^80,109 T. vitulorum, an ascarid that infects cattle, may also contribute to lung lesions during larval migration, especially in young calves. ²⁹ The migration of A. suum larvae through the lungs may result in IP and BrIntP, with hemorrhage, emphysema, and edema that manifests clinically as dyspnea, coughing, and, in severe cases, death.^{80,107,108,156} The clinical impact of A. suum in cattle ranges from mild respiratory signs to fatal pulmonary disease, depending on the infection burden and the host immune response. ⁶⁴ The diagnosis is based on compatible clinical signs, and gross and histologic lesions.

Algorithm and differential diagnoses for infectious and non-infectious causes of IP and BrIntP

An algorithm is used in medical textbooks and scientific articles to describe a clinical strategy designed to help medical professionals in clinical decision-making. ¹⁵¹ Our algorithm can guide a diagnostic investigation by organizing relevant information obtained from the postmortem examination and ancillary laboratory tests. It allows veterinarians to 1) reduce possible causes during the gross examination and 2) attempt to confirm the cause of IP or BrIntP. We understand that other approaches can be valid and that the diagnosis of any disease ultimately relies on the professional knowledge, experience, and judgment of the attending veterinary professional.

The BRDC is often a multifactorial syndrome in which management, environmental, animal, and pathogen factors interplay to cause disease. Moreover, the BRDC can also be multi-agent, with more than one respiratory pathogen involved. ⁹⁶ As with any diagnostic investigation, it is essential to begin with the collection of a concise and relevant clinical history that includes information about management, environment, weather, animal category, nutrition and nutritional condition, vaccination status, and clinical signs. ³⁷ Many of the clinical signs in animals with IP and BrIntP overlap and, therefore, are nonspecific. Clinical signs include decreased feed intake, inappetence, lethargy, ocular or nasal secretions, coughing, dyspnea, and, occasionally, sudden death. A comprehensive analysis of the epidemiology and clinical aspects of the different causes of the BRDC in cattle is outside our scope. Some aspects of the clinical history and epidemiology of the different causes were discussed earlier in this manuscript under each specific cause and are available elsewhere in the scientific literature.^19,27,31,129

We begin the algorithm listing the most common causes of IP and BrIntP, followed by their possible gross lesion distributions and the differential diagnoses to consider at the gross level ( Fig. 1 ). Next, we describe the histopathology of IP and BrIntP and some of the specific features (or lack thereof) of the most common infectious and non-infectious causes. Finally, we discuss a range of ancillary laboratory tests to help confirm the cause involved.

Figure 1.

Algorithm for the determination of infectious and non-infectious causes of interstitial pneumonia (IP) and bronchointerstitial pneumonia (BrIntP) in cattle. Beginning with the possible causes of IP and BrIntP (red box, top center), the solid arrows lead to their possible gross presentations, and then to the histopathology and main ancillary laboratory tests available for disease confirmation (blue boxes). The dashed arrows lead to the gross differential diagnoses (orange boxes) for the diffuse and cranioventral distributions of IP and/or BrIntP. BALT = bronchiolar-associated lymphoid tissue; BoHV1 = bovine alphaherpesvirus 1; BPIV3 = bovine parainfluenza virus 3; BRSV = bovine respiratory syncytial virus; D. viviparus = Dictyocaulus viviparus; DFA = direct fluorescent antibody; IHC = immunohistochemistry; ISH = in situ hybridization; MALDI-TOF MS = matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry.

Causes of IP and BrIntP

Viruses, ingested or inhaled toxins, bacterial septicemia or sepsis, and lungworms (chiefly D. viviparus) are important causes of diffuse IP in cattle. Viruses and, less frequently, some ingested toxins, also damage bronchial and bronchiolar epithelium in addition to the alveolar septa and, therefore, produce BrIntP. BrIntP is a microscopic term that can appear grossly as a diffuse pattern of IP or as a cranioventral distribution of consolidation, similar to that of bronchopneumonia.

Gross examination and gross differential diagnoses

Diffuse distribution of IP and BrIntP

We use the term “diffuse” here to indicate that all lung lobes, or most of the lungs, are affected. If the diffuse change is uniform, we use the term “diffuse-uniform” change. When the diffuse change is heterogeneous, as when affected lobules are mixed with less or non-affected lobules (“checkerboard” appearance), we use the term “diffuse-lobular” change. ⁹⁶

IP can have a diffuse-uniform ( Figs. 2–4 ) or diffuse-lobular ( Fig. 5 ) gross pattern. Lungs with a diffuse pattern of IP are non-collapsing, expanded (often with rib impressions on the pleural surface), possibly overinflated, and heavy, with a rubbery consistency. Compared with normal lungs, they may appear dark-pink or red. The interlobular septa are variably expanded by edema and/or emphysema. Foam may be present in the trachea and major airways, and petechiae and ecchymoses may occur on pleural surfaces. Pulmonary emphysema in cattle can be severe, but it is a nonspecific lesion that must be interpreted with caution. Emphysema can be an agonal finding or part of a clinically significant disease process, such as IP, BrIntP, ²⁷ or other cause of dyspnea.

Figures 2–7.

Gross distribution of interstitial (IP) and bronchointerstitial (BrIntP) pneumonia. Figure 2. Bovine respiratory syncytial virus, diffuse uniform distribution of BrIntP. Asterisk indicates an area of severe emphysema. (Image by Dr. Nicolas Streitenberger, University of California, Davis, USA). Figure 3. Salmonella spp. septicemia, diffuse uniform distribution of IP (Image by Dr. Germán Cantón, INTA Balcarce, Argentina). Figure 4 . Non-infectious interstitial pneumonia (presumptive fog fever), diffuse uniform distribution of IP. Asterisk indicates emphysema in the mediastinum. Figure 5. Dictyocaulus viviparus, prepatent stage, diffuse lobular distribution of IP or BrIntP (Image by Dr. Germán Cantón, INTA Balcarce, Argentina). Figure 6. Bovine respiratory syncytial virus, cranioventral distribution of BrIntP with normal dorsocaudal regions. Figure 7. Bronchopneumonia with interstitial pneumonia (BIP). Cranioventral consolidation and dark-red discoloration (bronchopneumonia), and caudodorsal diffuse lobular distribution of IP. Asterisk indicates area with interlobular emphysema (Image by Dr. Jeff Caswell, University of Guelph, Canada).

Viral respiratory pathogens, such as BRSV and BPIV3, pulmonary dictyocaulosis, septicemia or sepsis, and non-infectious (toxic) causes can have a diffuse pattern of IP. The gross pathology of toxic causes of IP may be indistinguishable from the diffuse pattern of infectious IP or BrIntP. Therefore, the clinical history is crucial when trying to rule in (or out) toxic IP at autopsy. A history of adult animals developing acute respiratory signs a few days after being moved from mature or dry pastures to lush and tender pastures suggests the possibility of acute bovine pulmonary emphysema and edema (fog fever).^26,138 Access or exposure to respiratory toxins—such as paraquat, 4-ipomeanol (from sweet potatoes), or toxic plants such as Brassica sp., Crofton weed, Crotalaria spp., and others—must raise the suspicion of a non-infectious cause of IP with diffuse alveolar damage.^{26,45,46,96,138} During the prepatent period of dictyocaulosis, the gross changes may be very similar to toxic IP or viral BrIntP given that larvae are not observed grossly and adults are not yet present in the airways. ¹²⁵ As dictyocaulosis progresses from the late-prepatent to the patent stage, adult nematodes can be found in airways, which is diagnostic for pulmonary dictyocaulosis.

Gross differential diagnoses of the diffuse gross pattern of IP include all causes of IP and BrIntP and any cause of pulmonary edema, congestion, and/or hemorrhage. Common causes of severe pulmonary edema include, but are not limited to, cardiac disease (left-sided heart failure, nutritional and toxic cardiomyopathies, cardiac histophilosis, cardiac blackleg, cardiotoxins, nutritional cardiomyopathy, bacterial valvular endocarditis, cardiac lymphoma), anaphylaxis, Clostridium perfringens type D enterotoxemia, downer syndrome, and others.^57,140 When pulmonary edema is accompanied by any combination of hydrothorax, hydropericardium, ascites, ventral subcutaneous edema, gallbladder wall edema, hepatomegaly, and/or a liver with a distinct reticulated acinar pattern (“nutmeg” liver) as the result of chronic passive congestion, it indicates right- and left-sided (global) heart failure.^36,140 Global heart failure can happen in toxic and nutritional cardiomyopathies, as well as in certain infectious causes of myocardial disease. Therefore, it is important to closely examine the heart and assess for both cardiac and extracardiac abnormalities commonly associated with left or global heart failure.^52,119 Gross lesions in the heart may not be obvious. Thus, representative sampling of the heart for histopathology is highly recommended when heart failure is suspected.

Anaphylactic shock or anaphylactoid reactions can cause severe acute respiratory distress with significant pulmonary edema. Accompanying laryngeal edema may help establish a presumptive diagnosis of anaphylaxis, but it is not always present and can be a nonspecific finding. A supportive clinical history, along with pulmonary edema and/or laryngeal edema, as well as ruling out other causes of disease, is very important when making a diagnosis of anaphylactic shock or anaphylactoid reactions. ²⁶

Cranioventral distribution of BrIntP

BrIntP caused by BRSV or BPIV3 can also have a cranioventral gross distribution, and the affected portion of lung can involve up to 60–70% of the pulmonary mass ( Fig. 6 ). ²⁷ Grossly, the lungs have a well-demarcated, cranioventral area of discoloration (often dark-pink to red), atelectasis, and a moderately firm consistency. A variable amount of foam can be present in the trachea and airways. Lungs with the cranioventral distribution of BrIntP can be accompanied by expanded caudodorsal areas that fail to collapse and can be edematous and rubbery. ²⁶ Typically, pleural fibrin or suppurative exudate in bronchi and bronchioles are not significant features, unless there is secondary bacterial infection. Lungs with a cranioventral distribution of BrIntP may be difficult to differentiate grossly from lungs suffering from bacterial bronchopneumonia. Lungs with bacterial bronchopneumonia are typically more swollen and firmer than those with cranioventral viral BrIntP caused by BRSV or BPIV3. ²⁶ In addition, lungs with bacterial bronchopneumonia often have fibrinous or fibrinosuppurative exudate on the pleural surface, whereas lungs with IP or BrIntP do not. ⁹⁶ Even when the distribution, texture, color, and type of exudate grossly suggest bacterial bronchopneumonia, histopathology can help to determine if there is an underlying concurrent viral infection—evidenced by microscopic lesions of BrIntP admixed with those of classic bacterial bronchopneumonia.

Bronchopneumonia with IP (BIP) has been reported in feedlot cattle in Canada. Grossly, BIP is a combination of 2 classic gross patterns, cranioventral bronchopneumonia and caudodorsal IP. In BIP, cranioventral regions are discolored and consolidated and caudodorsal regions are expanded, overinflated, and have interlobular edema and emphysema ( Fig. 7 ). The cause and pathogenesis of BIP are not completely understood.^72,73

Histopathology

We recommend performing histopathology whenever possible. Lung sampling for histopathology should include 4–6 representative pieces of lung. In lungs with a diffuse-uniform distribution of IP, obtaining 2 pieces from each cranial lung lobe and 2 pieces from each caudodorsal lung lobes should be sufficient in most instances. In lungs with a diffuse-lobular distribution of IP or in the cranioventral distribution of BrIntP, in which there are grossly normal areas, additional sections are often helpful (e.g., from the juncture between normal and abnormal lung and from the grossly normal area).

The diffuse alveolar damage that characterizes IP in cattle is divided into 3 overlapping phases: 1) an acute or exudative phase, 2) an organizing subacute (or proliferative) phase, and 3) a fibrotic chronic phase. ²³ In the acute or exudative phase, septa are congested and alveoli are filled with protein-rich edema fluid, fibrin, and variable numbers of neutrophils and macrophages; aggregates of fibrin, other serum proteins, and cell debris (hyaline membranes) cover damaged alveolar septa. In the subacute stage, type II pneumocytes proliferate to replace injured type I pneumocytes, repopulate the alveolar epithelium, and secrete new basement membrane. This change can be observed 2–3 d after injury and can be extensive by day 6. Cattle that survive the acute and subacute phases of IP or BrIntP may develop chronic IP, in which interstitial fibrosis of the alveolar wall can start as early as 3–5 d post injury and be well developed in 14 d.^23,26 Mononuclear interstitial inflammation, type II pneumocyte hyperplasia, and obliterative bronchiolitis (only in BrIntP) can all be present in the chronic stages.^26,72,96 In this stage, the lesions are nonspecific and, therefore, establishing the cause may not be possible based on microscopic lesions.

In the early stages of disease, certain characteristic histologic lesions can help differentiate among viral BrIntP, toxic IP, parasitic IP, bacterial bronchopneumonia, and bacterial septicemia or sepsis. BrIntP with epithelial syncytia formation, and eosinophilic ICIBs is characteristic of a viral infection, such as BRSV or BPIV3 ( Figs. 8–10 ). BPIV3 can also occasionally produce eosinophilic INIBs. However, it is important to note that the absence of syncytia and ICIBs or INIBs does not rule out BRSV or BPIV3 as possible causes, given that inclusion bodies can be absent in the subacute and chronic stages of the disease. Viral INIBs can be observed with BoAHV1 infection, which, although infrequent in the natural form of the disease, can also produce epithelial syncytia. If epithelial syncytia or viral inclusion bodies are not present, viral BrIntP may be impossible to distinguish from toxic causes of IP or BrIntP on histopathology. ²⁶ Subacute-to-chronic lesions in cases of viral or parasitic BrIntP include obliterative bronchiolitis ( Fig. 11 ), hyperplasia of bronchiolar-associated lymphoid tissue, pneumocyte type II hyperplasia, and fibrosis of peribronchial alveolar septa.

Figures 8–12.

Histology of viral bronchointerstitial (BrIntP) and non-infectious interstitial (IP) pneumonia. H&E. Figure 8. Bovine respiratory syncytial virus (BRSV) infection. Degeneration and necrosis of bronchiolar epithelium with formation of epithelial syncytia (arrowheads). Epithelial syncytia are also in the alveolar lumens (arrows). Alveolar septa are hypercellular; alveolar lumens are filled with edema, fibrin, erythrocytes, and inflammatory cells. Inset: epithelial syncytia with eosinophilic intracytoplasmic viral inclusion bodies surrounded by neutrophils. Figure 9. Bovine parainfluenza virus 3 (BPIV3) infection. Bronchiolar degeneration and necrosis, with epithelial attenuation (arrowheads), lymphoplasmacytic infiltration of peribronchiolar and perivascular interstitium, type II pneumocyte hyperplasia (arrow), and intra-alveolar hemorrhage and fibrin. Figure 10. BPIV3 infection. Marked type II pneumocyte hyperplasia and numerous eosinophilic intracytoplasmic (arrow) and intranuclear (arrowheads) viral inclusion bodies. Figure 11. BRSV infection. The lumen of a bronchiole is partially obliterated by a fibrovascular proliferation (asterisk) that extends from the bronchiolar wall and is partially covered by flattened epithelium (arrowhead). Figure 12. Non-infectious interstitial pneumonia (presumptive fog fever). In the alveolar septa, type I pneumocytes are damaged, and type II pneumocyte hyperplasia is evident (arrows). Hyaline membranes (arrowheads) line alveoli; macrophages, cellular debris, and edema are within the alveolar lumens.

IP caused by ingested toxins, such as 3MI, 4-ipomeanol, perilla ketone, Brassica spp., and C. mucronata, has diffuse alveolar damage and can have exudative and proliferative stages. There is damage to type I pneumocytes, formation of hyaline membranes, proliferation of type II pneumocytes, edema, and emphysema ( Fig. 12 ). Damage to the bronchial or bronchiolar epithelium is usually not present in the natural form of 3MI intoxication, but can be present after experimental administration of 3MI. Intoxication with 4-ipomeanol may or may not induce injury to airway epithelium. ²⁶ Inhalation or ingestion of paraquat, a highly toxic herbicide, can cause acute diffuse alveolar damage if inhaled or ingested. Severe edema and hemorrhage can be the predominant lesions in acute poisoning from exposure to a high dose of paraquat. ²⁶

In cases of parasitic IP caused by D. viviparus, the lesions vary according to the stage of infection, the immune status of the host, and the number of invading parasite larvae. During the prepatent period in naïve animals, findings include eosinophilic bronchiolitis and/or alveolitis with fibrin or hyaline membranes, degeneration, attenuation and necrosis of bronchiolar epithelium, and larvae in bronchioles and alveoli ( Fig. 13 ). In the patent period, bronchiolar hyperplasia and metaplasia may be observed. The inflammation targets adult worms in the bronchi and also eggs and L1 larvae that have been aspirated into the alveoli. It is important to highlight that some infections may be missed if only the trachea and large cranial bronchi are examined or sampled, because lesions and parasites are frequently found only in the small branches of the caudal bronchi. Emphysema can be present and may be severe when obstructive bronchiolitis is evident. During the recovery period, adult worms are progressively eliminated from the airways.^26,125,127

Figures 13–18.

Histology of parasitic and bacterial causes of interstitial pneumonia (IP). Figure 13. Dictyocaulus viviparus IP. Intraalveolar nematode larva (arrowhead), hypercellular alveolar septa, eosinophils and neutrophils in alveolar lumens (Image by Dr. Germán Cantón, INTA Balcarce, Argentina). Figure 14. E. coli septicemia, acute stage. Low magnification of marked diffuse hyperemia of alveolar septa and intraalveolar acute hemorrhage. Figure 15. E. coli septicemia, acute stage. Higher magnification of clumps of fibrin in alveolar lumens (arrowhead), alveolar septal hyperemia, and intra-alveolar acute hemorrhage. Figure 16. E. coli septicemia, acute stage. Clusters of degenerate and viable neutrophils within alveoli, alveolar septa congestion, and acute intra-alveolar hemorrhage. Figure 17. E. coli septicemia, acute stage. Alveolar septa are hypercellular, with evidence of intravascular (boxed area) and intra-alveolar bacteria (arrowhead). Inset: higher magnification of boxed area showing intracapillary bacteria (arrowhead). Figure 18. Salmonella Dublin septicemia, acute stage. Alveolar septa are hypercellular, with intra-alveolar edema and fibrin. Asterisks = normal bronchioles. Inset: higher magnification of thrombosis.

Septicemic interstitial lung injury can manifest histologically in a variety of ways, as described above ( Figs. 14–18 ). Key features for the diagnosis of IP resulting from bacterial septicemia or sepsis include marked congestion of alveolar septa, intraalveolar edema and hemorrhage, hypercellular alveolar septa, neutrophils, small venule and capillary thrombi, and a monomorphic population of bacteria within blood vessels and/or within alveolar lumens.^23,26 The absence of bacterial colonies in histologic sections does not exclude the possibility of septicemia, and the presence of postmortem bacteria in the lungs can complicate interpretation. Large bacilli or mixed bacteria in histologic sections of the lungs with moderate-to-advanced autolysis should raise the suspicion of postmortem bacterial invasion. Necrotizing bronchitis and bronchiolitis, a distinguishing lesion of viral BrIntP, is typically not a feature of bacterial IP or sepsis.

Ancillary laboratory tests

Laboratory tests are now more widely used to determine the exact cause(s) of the BRDC ( Table 1 ), especially given that treatment decisions involve group therapy and, in the environments of increased antimicrobial resistance and of antimicrobial use restrictions, mass medicating should be evidence-based. Laboratory tests can be performed to confirm acute disease or carrier state (virus isolation, PCR, IHC, bacterial cultures, and others) or to evaluate disease prevalence, herd exposure, or response to vaccination programs (serology).

Table 1.

Ancillary laboratory tests, sample type, pathogens, and main uses for the diagnosis of infectious causes of IP and BrIntP in cattle.

Test type	Sample type	Pathogen	Use
Histopathology	Lung	NA	Characterization of lung lesions
PCR	Nasal swab, BAL, lung tissue	BRSV, BoAHV1/5, BPIV3, BVDV, BCoV, IDV, MCFV, bacteria	Confirmation of active infection. Detection of viral or bacterial nucleic acids (RNA, DNA)
Virus isolation	Nasal swab, BAL, lung tissue	BRSV, BoAHV1/5, BPIV3, BVDV, BCoV, IDV	Confirmation of active infection
IHC	FFPE tissues	BRSV BoAHV1/5, BPIV3, BVDV, BCoV, IDV, some bacteria	Confirmation of active infection; detection of antigens in tissue; localization of viral or bacterial pathogens within the lesions
Serology	Serum or milk	BRSV, BoAHV1/5, BPIV3, BVDV, BCoV, IDV	Evaluation of disease prevalence, herd exposure, or response to vaccination programs; detection of antibodies; seroconversion can be used to detect active infection
Bacterial culture	Tissue, BAL	Salmonella spp., E. coli, others	Confirmation of active infection; bacterial isolation and identification
Baermann technique	Feces	Dictyocaulus viviparus	Confirmation of active infection; detection of first-stage larvae in feces

BAL = bronchoalveolar lavage; BoAHV 1/5 = bovine alphaherpesvirus 1 and 5; BCoV = bovine coronavirus; BPIV3 = bovine parainfluenza virus 3; BRSV = bovine respiratory syncytial virus; BVDV = bovine viral diarrhea virus; FFPE = formalin-fixed, paraffin-embedded; IDV = influenza D virus; IHC = immunohistochemistry; MCFV = malignant catarrhal fever virus; NA = not applicable (histology is used to characterize lesions but not for pathogen detection).

PCR tests are widely used for the detection of viral pathogens and some bacterial pathogens, such as Salmonella spp. PCR tests identify nucleic acids of pathogens and have high sensitivity. Many veterinary diagnostic laboratories offer PCR tests for individual pathogens or as part of a BRDC panel. PCR testing is commonly used for the detection of BoAHV1, BPIV3, BRSV, BVDV, BCoV, and the MCFV group. However, given that pathogen-specific primers are needed for PCR, the targeted virus must be suspected beforehand. If animals were recently vaccinated with live-virus vaccines, vaccine-associated nucleic acids can be detected by PCR testing. ¹²⁹ Another important consideration is that a positive PCR result for a given pathogen does not necessarily indicate a causal relationship with disease. Many animals harbor pathogens that may not cause clinical disease. A negative PCR result does not exclude the possibility of a viral cause, especially in the later stages of disease, as the virus may no longer be detectable in tissue samples.

Virus isolation has been largely replaced by faster and more affordable molecular methods, such as PCR, rtPCR, and IHC, in most diagnostic laboratories. ³⁵ However, in some cases (e.g., BoAHV1), virus isolation allows identification of novel strains that can then be archived. Virus isolation is not recommended for the detection of BRSV, because the immune response generated by the host interferes with the test and the virus is easily inactivated in transport. ²⁶

IHC is a useful tool that is available in some diagnostic laboratories for routine testing. Antibodies are used to detect antigens of specific pathogens in FFPE tissues. IHC is particularly useful because it allows localization of pathogens within the lesions, which strengthens causality and offers insights into disease pathogenesis. ¹⁷³

Serologic tests are used to detect antibodies and, therefore, provide indirect evidence of infection. They are especially useful for monitoring viral circulation at the population level and to evaluate the response to, and protection conferred by, vaccination programs. However, they are generally less useful for the diagnosis of acute disease, given that this typically requires paired samples collected in the acute and convalescent phases, with a 2–3-wk interval, significantly delaying the diagnosis. Moreover, serologic interpretation of respiratory diseases in cattle can be very difficult, as vaccination procedures and timing of sample collection can distort test results. ³⁵ To enhance the relevance of serology results for herd health assessments, multiple animals should be sampled.^27,129

Bacterial cultures can detect a broad range of viable bacterial pathogens and allow for antimicrobial susceptibility testing when required. ¹²⁹ When trying to confirm IP resulting from bacterial septicemia, submitting lung and an additional sample from another organ, tissue, or fluid can increase the chances of identifying the bacterium involved and improving test interpretation. Isolation or detection of the same microorganism from 2 different sources increases diagnostic confidence. Fresh postmortem specimens and clean, appropriate sample collection during the autopsy will make culture results more accurate and easier to interpret. Two important considerations when attempting bacterial cultures are 1) prior administration of antimicrobials, which can result in negative bacterial cultures, and 2) moderate-to-advanced postmortem decomposition, which can result in isolation of mixed bacteria because of postmortem bacterial invasion. The isolation of mixed bacteria from a specific organ can be interpreted as postmortem contamination, but it is possible that the bacterium involved in the septicemia is among those that are isolated. When no or only mild, nonspecific gross or histologic lesions are present, a pure culture of a single bacterial species—especially in large numbers and/or from more than one organ, tissue, or fluid—may be the best tool to confirm a presumptive diagnosis of IP caused by septicemia. A single, positive bacterial culture from the lung of a freshly dead animal can determine the cause with acceptable confidence when the clinical history, gross and histopathology support the diagnosis.

MALDI-TOF MS testing has become increasingly popular in clinical microbiology laboratories in the last decade. Once a bacterial isolate is obtained, MALDI-TOF MS can identify these isolates at least 24 h faster than the conventional biochemical identification systems. ²⁰ The use of MALDI-TOF MS directly on clinical samples has been proposed and may be available in the future.^20,166

Next-generation (NGS) and third-generation (TGS) sequencing technology have become available for diagnostic purposes, especially in resource-rich countries. These advanced molecular techniques enable high-throughput analysis of nucleic acids, which allows for simultaneous testing of a vast array of infectious agents. In the context of BRDC diagnosis, these technologies help identify known, emerging, and poorly characterized potential respiratory pathogens, allowing for strain typing and characterization of complex microbial communities. However, NGS and TGS are not widely available for routine diagnostic purposes because of cost and technical limitations.^{111,129,159,178}

No standard or readily available laboratory tests exist to detect exposure to, or confirm intoxication by, 3MI, 4-ipomeanol, and toxic plants associated with IP or BrIntP. Therefore, the diagnosis relies on clinical history, signs, and compatible gross and microscopic lesions. Paraquat can be detected in animal tissues and fluids, gastrointestinal contents, and feed by select veterinary toxicology laboratories.^26,138